Apolipoprotein A-I structure in high density lipoproteins

描述（由申请人提供）：尽管循环高密度脂蛋白（HDL）被认为可以预防心血管疾病，但我们对其结构的了解非常有限。此外，我们对主要的高密度脂蛋白，载脂蛋白（apo）A-I如何与其他蛋白质相互作用来决定高密度脂蛋白的功能了解得更少。该项目将测试apoA-I与人类血浆中常见的球形高密度脂蛋白颗粒中的其他高密度脂蛋白相关蛋白进行高度特异性接触的假设。在我们之前的工作中，我们使用交联化学和质谱法来生成重组颗粒中apoA-I的详细模型以及来自人血浆的“真实”HDL。尽管在大小和形状上有很大的不同，这些结构都有一个反平行的带状排列的主题。基于这些发现，我们的目标是使用互补结构技术进一步评估这些模型和其他模型，以及评估apoA-I与三种主要HDL成分相互作用的基础：载脂蛋白A-II、卵磷脂、胆固醇酰基转移酶（LCAT）和胆固醇酯转移蛋白（CETP）。具体目的是：1)利用新的双同位素交联技术和最先进的全原子和过程颗粒分子动力学（MD）技术，测试球面重构和天然血浆HDL中apoA-I基于trefoil的模型。2)利用交联和新的人类apoA-II细菌表达系统来确定apoA-I和apoA-II之间的分子相互作用，从而获得含有这两种蛋白的天然HDL颗粒的第一个模型。3)通过验证LCAT通过位点定向诱变和交联进入apoA-I结构中的分子孔的概念，确定apoA-I与LCAT之间的分子相互作用。我们还将确定CETP是否特异性结合apoA-I并确定它们的接触点。这种方法独特地将新的实验技术与最先进的MD方法结合在一起，这将扩大我们对这些神秘粒子的结构理解。apoa - 1的结构无疑调节HDL代谢。因此，对其结构及其与其他蛋白质的相互作用的分子理解，特别是那些正在作为药物靶点探索的蛋白质，如LCAT和CETP，对于设计利用HDL的逆向胆固醇转运和抗炎作用的新疗法至关重要。

DESCRIPTION (provided by applicant): Although circulating high density lipoproteins (HDL) are considered protective from cardiovascular disease, we have a remarkably limited understanding of their structure. Furthermore, we understand even less about how the major HDL protein, apolipoprotein (apo)A-I, interacts with other proteins to dictate HDL function. This project will test the hypothesis that apoA-I makes highly specific contacts with it and other HDL associated proteins in spherical HDL particles commonly found in human plasma. In our previous work, we used cross- linking chemistry and mass spectrometry to generate detailed models of apoA-I in reconstituted particles as well as "real" HDL from human plasma. Despite substantial differences in size and shape, these structures all shared the theme of an antiparallel belt-like arrangement. Building on these discoveries, our goal is to further evaluate these and other models using complementary structural techniques as well as evaluate the basis of apoA-I's interactions with three major HDL components: apolipoprotein A-II, lecithin:cholesterol acyl transferase (LCAT), and cholesteryl ester transfer protein (CETP). The specific aims are: 1) To test the Trefoil-based models of apoA-I in spherical reconstituted and native plasma HDL using new dual isotope cross-linking techniques and state-of-the-art all-atom and course grained molecular dynamics (MD) techniques. 2) To determine the molecular interactions between apoA-I and apoA-II using cross-linking and a new human apoA-II bacterial expression system to derive the first models of native HDL particles containing both proteins. 3) To determine the molecular interaction between apoA-I and LCAT by testing the concept that LCAT accesses a molecular pore in the apoA-I structure using site-directed mutagenesis and cross-linking. We will also determine if CETP specifically binds apoA-I and identify their points o contact. This approach uniquely intertwines new experimental techniques with state-of-the-art MD approaches that will magnify our structural understanding of these enigmatic particles. The structure of apoA-I undoubtedly modulates HDL metabolism. Thus, a molecular understanding of its structure and its interactions with other proteins, particularly those being explored as dru targets such as LCAT and CETP, is critical for the design of new therapies exploiting reverse cholesterol transport and the anti-inflammatory roles of HDL.